Biology or chemistry performed on the micro-scale, involving the reaction and subsequent analysis of quantities of reagents or analytes on the order of microliter or sub-microliter amounts, is an increasingly important aspect in the development of new therapeutic agents in the pharmaceutical and other industries. Both to conserve limited samples, reagents, or precious chemical compounds and to increase high-throughput screening capacity, clinical, biological or pharmaceutical researchers often employ reduced-volume assays to detect disease, study biochemical functions, or discover drugs. Several difficulties arise, however, as assay volumes are reduced to microliter and sub-microliter amounts. First, very low volumes of aqueous solutions tend to evaporate rapidly, which causes the concentrations of reagents in solution to change as the amount of water changes. As a consequence, small volume assays often fail to give consistent, accurate results. Second, signals generated from small-volume assays can be imperceptibly low, which may often generate false negative or positive results. Since long exposure times are required for detection of low signal levels by charge-coupled devices (CCD), the ability to process assays quickly with high-throughput capacity is compromised.
The existing technology has relied principally on 96-, 384, or 1536-well microtiter plates containing assay quantities between approximately 0.5 microliter and 0.5 milliliter of liquid compound per well, or involves chemical reactions and analysis in wells disposed with single openings on flat two dimensional surfaces such as glass slides or silicon chips. Unfortunately, these techniques appear to be of moderate success for addressing the issue of evaporation, and of even more limited effectiveness for improving signal detection. A microplate format is not properly configured for efficient collection or direction of an optical signal. The fluorescent signal emitted from chromophores or fluorephores used in biological, biochemical, or chemical assay reactions often is scattered.
Existing microtiter plates passively allow optical signal generated from an assay to escape from the top of the well at random angles with a small percentage of the generated signal being captured by a detector. Fluorescence based assays typically utilize wells with black, light absorbing walls that keep the background from the plate low but have the disadvantage of absorbing the majority of the assay signal. As signal intensity becomes of greater and greater importance as assay volume decreases, these issues become more and more inhibitory to plate performance. Previously, others have tried to address electromagnetic detection of small sample volumes, in particular as relating to capillary electrophoresis systems. Pentoney, Jr. et al., in U.S. Pat. No. 5,675,155, details a system that uses co-planar side-by-side capillaries for sequentially and repetitively scanning a plurality of sample volumes and detecting electromagnetic radiation emitted from each of the sample volumes. Each of the capillaries, oriented in a perpendicular fashion to a mirror reflector, constitutes a single-point radiant source. The system also includes a means for moving and adjusting the mirror's position. This design, however, can not be adapted easily for use with a conventional microplate, and is not compatible with current robotically automated assay processing equipment. The spatial orientation and configuration of the capillaries makes integration with a microplate difficult. That is, one can not merely insert the capillary into a microplate and fill the capillaries. Rather, for the Petoney device to work efficiently with microplates, it appears to require repositioning and various adjustments.
In view of these disadvantages, a new device and method for performing small-volume assays is needed. The new device and method both should be cost-efficient to use and manufacture, efficient for detection, and have high-throughput capability to enable simultaneous mass-volume processing. The present invention can both solve the aforementioned technical problems and satisfies the economic and efficiency needs.